BIP - Production of polyclonal antisera to manganese superoxide dismutase expressed in downy mildew resistant pearl millet and its application for immunodiagnosis

Financial support: Indian Council of Agricultural Research, New Delhi and Council for Scientific and Industrial Research, New Delhi, India.

Keywords: DIBA, downy mildew, ELISA, immunofluorescence, manganese superoxide dismutase, pearl millet, Sclerospora graminicola, Western blot.

Downy mildew of pearl millet (Pennisetum glaucum L. (R.)Br.( incited by S. graminicola (Sacc.) Schroet. is a devastating disease. The disease is systemic and the pathogen is an obligate biotroph. Its economic significance has become more accentuated than ever before, especially in relation to the high yielding genotypes (Shetty, 1990). Considerable progress has been made in understanding the interaction between the downy mildew pathogen and pearl millet. Particularly striking has been the information gained at the histological and biochemical levels (Shetty et al. 1998). The role of the enzyme superoxide dismutase in imparting resistance to downy mildew in pearl millet has been established by the authors (Babitha et al. 2002a).

Reactive O₂ species (ROS) are produced in both unstressed and stressed cells. Plants have well-developed defence systems against ROS, involving both limiting the formation of ROS as well as instituting its removal (Alscher et al. 2002). Within a cell, the superoxide dismutases (SODs) constitute the first line of defence against ROS (Alscher et al. 2002). Superoxide dismutases (SODs; EC 1.15.1.1) are a family of metalloenzymes that catalyze the disproportionation of superoxide (O₂^.-) radicals, and they play an important role in protecting cells against the toxic effects of superoxide radicals produced in different cellular loci (Halliwell and Gutteridge, 2000). It has been extensively reported in the literature that the response of SOD activity and other antioxidative enzymes to oxidative stress varies according to the environmental conditions, plant tissue, stage of development, etc (Alscher et al. 2002). Three classes of SODs, differing in the metals at their catalytic active site, are known in plants. The CuZnSODs are localized in the cytosol, chloroplasts, nucleus, and apoplast; the Mn-SODs in the mitochondria and peroxisomes; and the FeSODs in the chloroplasts (Ogawa et al. 1996). Both O₂^.-and H₂O₂ have been shown to act directly or indirectly in plant defence and signal transduction (Vranova et al. 2002). The conversion of O₂^.-and H₂O₂ to OH^., catalyzed by transition metals (Haber-Weiss reaction), accounts for the severe toxicity of ROS in plants (Wojtaszek, 1997).

Activated oxygen or oxygen-free radical mediated damage to plants has been implicated in many plant stress situations. The enhanced SOD activity might increase oxidative stress due to increased production of H₂O₂(Tenhaken et al. 1995). Increased activity of the enzyme has been reported to induce cell dysfunction and death. Moreover H₂O₂ is implicated in hypersensitive cell death, thus limiting the spread of cell death by induction of cell protectant genes in surrounding cells. H₂O₂ also inhibits the growth and viability of diverse microbial pathogens (Wu et al. 1995). The oxidative potential of H₂O₂ also contributes to plant cell wall strengthening during plant-pathogen interactions through peroxidase-mediated cross-linking of proline-rich structural proteins and phytoalexin biosynthesis during oxidative burst. Manganese-containing superoxide dismutases have been characterized from a wide range of organisms including bacteria, algae, fungi, and animals (Bannister et al. 1987), and also from several higher plants (Streller et al. 1994; Kroniger et al. 1995). In eukaryotic cells, Mn-SODs have been found to be localized mainly in mitochondria from different organisms (Halliwell and Gutteridge, 2000). Mn-SODs are chiefly present in mitochondria (Halliwell and Gutteridge, 2000), but also occur in different types of peroxisomes (Corpas et al. 1998). Mn-SODs efficiently remove superoxide radicals, but produce H₂O₂as a by-product of their catalytic reaction (Fridovich, 1995). High concentrations of H₂O₂ are dangerous to the plant cell, but at low concentrations this metabolite also acts as a diffusible signaling molecule in signal transduction pathways that lead to the activation of gene expression (Grant and Loake, 2000).

The present investigation was aimed at production of polyclonal antibody against Mn-SOD. The antibody was used to examine the Mn-SOD protein in downy mildew defense through ELISA, DIBA, Western blot and immunofluorescence. Comparision of Mn-SOD activity with Mn-SOD protein amount which was determined immunologically indicates that the activity of the enzyme is increased in the downy mildew resistant pearl millet after inoculation with the pathogen S. graminicola.

The seed samples were collected from ICRISAT, Hyderabad and All India Co-ordinated Pearl Millet Improvement Project (AICPMIP), Jodhpur, India. Two-day old seedlings were inoculated with the pathogen S. graminicola. The enzyme was extracted at different time intervals by following the method of Dhindsa et al. (1981). The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) using the method of Beauchamp and Fridovich (1971). Differential induction of superoxide dismutase (SOD) in downy mildew-resistant and -susceptible genotypes of pearl millet (Pennisetum glaucum) was observed on inoculation with Sclerospora graminicola (Babitha et al. 2002a). SOD activity was studied in resistant (IP18292) and susceptible (23B) pearl millet seedlings inoculated with S. graminicola. SOD activity increased by 2 x 3-fold in resistant seedlings upon inoculation. Native PAGE analysis showed four isozymes of SOD, three of which (SOD-1, -2 and -4) were Cu/Zn-SOD, whereas isozyme SOD-3 was Mn-SOD whose intensity increased in the resistant genotype upon inoculation. The enzyme Mn-SOD was purified from inoculated resistant pearl millet seedlings by homogenization, ammonium sulphate precipitation, anion exchange chromatography and gel-filtration chromatography followed by SDS-PAGE (Babitha et al. 2002b). Mn-SOD was purified 73-fold from pearl millet. Electrophoresis revealed a single band of SOD activity corresponding to the purified enzyme.

This was used as antigen for antiserum production and to test the serological relationships of polyclonal antibody with pure Mn-SOD and crude SOD extract of different pearl millet genotypes with varying degrees of resistance to downy mildew disease.

This study determines the validity of utilizing the various immunological assays for determining Mn-SOD reactivity in pearl millet seedlings. To accomplish this Mn-SOD was purified from inoculated resistant pearl millet seedlings. Polyclonal antibody against Mn-SOD was obtained after intramuscular injections. The purified enzyme was utilized to develop sensitive and reproducible immunoassays, the specificity of which was tested by various serological tests. The present paper describes the attempts made and strategy employed to obtain specific polyclonal antibody against Mn-SOD and to develop a sensitive, rapid and specific ELISA to employ in routine downy mildew disease screening technique among the various pearl millet genotypes with varying degree of susceptibility/resistance. The antiserum was found to be extremely immunogenic as assessed by the titration of the immune sera. The ELISA developed was shown to be very sensitive with a titre of 1:20000. The antibody reacted more strongly with pure Mn-SOD than with crude SOD extract as shown by ELISA and DIBA. The SOD reactivity was significantly higher in inoculated pearl millet seedlings as compared to the uninoculated pearl millet resistant seedlings as detected by ELISA and DIBA. Tissue-specific expression as shown by ELISA and DIBA revealed a high SOD reactivity in inoculated root of resistant seedlings. The data from ELISA and DIBA suggest a correlation between SOD reactivity, SOD activity and degree of resistance of pearl millet genotype. To investigate the potential of immunological systems for identification of SOD, antiserum has been produced which shows specificity towards the antigen used but also confirms the existence of variation between genotypes of pearl millet. Additional serological tests like western blot and immunofluorescence confirmed the results. Western blots of pearl millet seedlings revealed a single band, corresponding to the 35 kDa purified protein. It showed an increased accumulation of SOD protein in the resistant seedlings after inoculation. Recombinantly expressed mitochondrial Mn-SOD was used to raise polyclonal antibodies, which cross-reacted with Mn-SOD in peroxisomes purified from pea leaves. Western blot assays of crude extracts with the antibodies to pea mitochondrial Mn-SOD showed that the levels of total Mn-SOD protein gradually increased with leaf senescence.

Anti-SOD antiserum, when used in immunofluorescence experiments revealed two major locations for the antigen observable in fluorescence micrographs; the vascular bundle and the epidermis. The absence of specific fluorescence in the control tests indicated that the resultant staining depended only on antibody-antigen binding sites. EM immunochemistry was used to distinguish mitochondria and peroxisomal Mn-SOD in senescent leaves. Increased Mn-SOD labeling of perxisomes did not change with senescence (del Rio et al. 2003). The serological procedures used in these studies were sensitive enough to detect SOD in pearl millet genotypes. In summary, the data presented in this paper indicate that SOD can be detected immunologically in pearl millet and have sufficient specificity for simple identification purposes.

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